This is an application to renew a project that significantly involves undergraduate students in research on synaptic function at the vertebrate neuromuscular junction (NMJ). For many years, experiments performed on the NMJ, the chemical synapse between a motor nerve and muscle, provided much of our basic understanding of synapses. As this knowledge grew and as techniques became more sophisticated, most neuroscientists turned their attention to synapses in the CNS. Only a relatively few have continued studying the one synapse that absolutely must function properly for an animal to survive. Despite its apparent simplicity, the NMJ uses a complicated set of pathways that modulate synaptic transmission. Since all of these have counterparts in the CNS, exploration of NMJ modulatory mechanisms will not only extend our understanding of the NMJ per se but will also provide useful insights about synapses in general. The research proposed in this grant will explore three features of synaptic modulation. First, it seeks to understand the roe of endocannabinoids (eCBS) at the vertebrate NMJ. This knowledge is critical as efforts are currently underway to develop peripherally active eCB antagonists to treat obesity and its sequelae. As at many central synapses, the eCB 2-arachidonylglycerol (2- AG) acts as a retrograde signal: it is produced in the postsynaptic cell, in this case the muscle, and inhibits neurotransmitter release by activating CB1 receptors on the presynaptic nerve terminal. Experiments are planned to better understand this process at lizard NMJs, where it was first discovered, and at the mouse NMJ, which will be the first study of its kind at a mammalian NMJ. The second goal of this project is to contribute to the ongoing debate over whether the NMJ is a tripartite synapse. Just as glial cells are known to play a crucial role in the function and plastiity of synapses in the CNS (the dysfunction of which contribute to neurological and psychiatric illness), there is growing evidence that perisynaptic Schwann cells (PSC) play an analogous if not identical role at the NMJ. Using a novel technique for ablating (i.e. killing) PSCs at the NMJ that spares the nerve terminal and muscle, we will test three hypotheses: (i) that cyclooxygenase-2 (COX-2), which converts 2- AG to a glycerol ester of prostaglandin E2 within PSCs, is essential for the delayed enhancement of neurotransmitter release triggered by the activation of muscarinic ACh receptors (mAChRs), (ii) that activation of mAChRs induces the transcription of COX-2 mRNA in PSCs and thereby increases levels of COX-2, and (iii) that NMJs missing all or some of their PSCs will lack certain types of synaptic plasticity. The final goal of the proposed research is to solve the mystery of glutamate at the NMJ. Although the NMJ is a cholinergic synapse, new evidence is accumulating suggesting that glutamate plays an important role; however, the source of glutamate and its function have remained unclear. We will test the hypothesis that N-acetylaspartylglutamate (NAAG) is the source of the glutamate and that the activation of glutamate receptors, by both glutamate and NAAG, modify synaptic transmission through their ability to control the synthesis of nitric oxide.